regulation of stem cell maintenance by the polycomb ... · henderson and jacobsen, 2007; reik et...

2433RESEARCH ARTICLE

INTRODUCTIONAll land plants are characterized by an alteration of two generations:the haploid gametophyte and the diploid sporophyte. In floweringplants, the sporophyte comprises complex organs including leafyshoots and flowers. Here, this phase dominates over the diminutiveand ephemeral gametophytic phase. The gametophytes of floweringplants, namely the pollen and female embryo sacs in whichfertilization occurs, are epiphytic to the diploid plant body. Incontrast to flowering plants, in bryophytes, the earliest diverginggroup in land plant evolution, the gametophytic generation isphotosynthetically active and dominates the epiphytic sporophyte(reviewed by Reski, 1998a). Consequently, bryophytes propagatethrough haploid spores, whereas flowering plants propagate viadiploid seeds. The last common ancestor of bryophytes andflowering plants was estimated to live around 500 million years ago(Zimmer et al., 2007), an evolutionary distance similar to thatbetween human and fish.

In recent years it has become evident that mechanisms for genesilencing play a role in regulating developmental programs. Ingeneral, silencing involves both nucleic acid-based mechanisms,such as small RNA molecules (Bartel, 2004; Jones-Rhoades et al.,2006; Zhang et al., 2007) or DNA methylation (Ginder et al., 2008;

Henderson and Jacobsen, 2007; Reik et al., 2001; Saurin et al.,2001), as well as histone-based modifications (Jenuwein and Allis,2001), such as methylation of lysine 27 on histone 3 (H3K27me3)(Lachner et al., 2003). Methylation of H3K27 is mediated by thePolycomb recruiting complex 2 (PRC2) (Czermin et al., 2002; Ketelet al., 2005; Muller et al., 2002; Nekrasov et al., 2005), alsodesignated the Polycomb group protein (PcG) complex. The PcGcomplex was first identified in Drosophila melanogaster (Jurgens,1985; Lewis, 1978) and subsequently in Caenorhabditis elegans(Holdeman et al., 1998), Homo sapiens (Chen et al., 1996;Denisenko and Bomsztyk, 1997), as well as in flowering plants(Goodrich et al., 1997; Grossniklaus et al., 1998; Ohad et al., 1999;Luo et al., 1999).

In A. thaliana genetic and biochemical analyses predict severalPcG-like PRC2 complexes (Goodrich et al., 1997; Grossniklaus etal., 1998; Ohad et al., 1999; Luo et al., 1999; Chanvivattana et al.,2004), some of which have been isolated and identified (De Luciaet al., 2008; Wood et al., 2006). All PcG complexes in A. thalianacomprise the WD40 motif-containing proteins FERTILIZATIONINDEPENDENT ENDOSPERM (FIE) and MULTICOPYSUPRESSOR OF IRA 1 (MSI1) (Kohler et al., 2003b; Ohad et al.,1999). In addition, each PcG complex is predicted to contain one ofthe three SET domain proteins CURLY LEAF (CLF), SWINGER(SWN) or MEDEA (MEA) (Chanvivattana et al., 2004; Luo et al.,1999; Katz et al., 2004; Yadegari et al., 2000). The SET domainprotein acts as the catalytic subunit and methylates H3K27me3(Czermin et al., 2002; Ketel et al., 2005; Muller et al., 2002;Nekrasov et al., 2005). Members of the SET domain PcG proteinsmay interact with one of the zinc-finger PcG proteins, includingEMBRYONIC FLOWER 2 (EMF2), VERNALIZATION 2(VRN2) or FERTILIZATION INDEPENDENT SEED 2 (FIS2), viathe VEFS domain (Chanvivattana et al., 2004).

Regulation of stem cell maintenance by the Polycombprotein FIE has been conserved during land plant evolutionAssaf Mosquna1, Aviva Katz1, Eva L. Decker2,4, Stefan A. Rensing3,4, Ralf Reski2,3,4 and Nir Ohad1,*

The Polycomb group (PcG) complex is involved in the epigenetic control of gene expression profiles. In flowering plants, PcGproteins regulate vegetative and reproductive programs. Epigenetically inherited states established in the gametophyte generationare maintained after fertilization in the sporophyte generation, having a profound influence on seed development. Thegametophyte size and phase dominance were dramatically reduced during angiosperm evolution, and have specialized inflowering plants to support the reproductive process. The moss Physcomitrella patens is an ideal organism in which to studyepigenetic processes during the gametophyte stage, as it possesses a dominant photosynthetic gametophytic haploid phase andefficient homologous recombination, allowing targeted gene replacement. We show that P. patens PcG protein FIE (PpFIE)accumulates in haploid meristematic cells and in cells that undergo fate transition during dedifferentiation programs in thegametophyte. In the absence of PpFIE, meristems overproliferate and are unable to develop leafy gametophytes or reach thereproductive phase. This aberrant phenotype might result from failure of the PcG complex to repress proliferation anddifferentiation of three-faced apical stem cells, which are designated to become lateral shoots. The PpFIE phenotype can bepartially rescued by FIE of Arabidopsis thaliana, a flowering plant that diverged >450 million years ago from bryophytes. PpFIE canpartially complement the A. thaliana fie mutant, illustrating functional conservation of the protein during evolution in regulatingthe differentiation of meristematic cells in gametophyte development, both in bryophytes and angiosperms. This mechanism washarnessed at the onset of the evolution of alternating generations, facilitating the establishment of sporophytic developmentalprograms.

KEY WORDS: Apical cell, Arabidopsis thaliana, BiFC, CLF, PcG complex, Physcomitrella patens, Protein-protein interaction

Development 136, 2433-2444 (2009) doi:10.1242/dev.035048

1Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel. 2PlantBiotechnology, Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104Freiburg, Germany. 3Freiburg Initiative for Systems Biology (FRISYS), University ofFreiburg, Schänzlestrasse 1, 79104 Freiburg, Germany. 4Centre for BiologicalSignalling Studies (bioss), University of Freiburg, Albertstrasse 19, 79104 Freiburg,Germany.

*Author for correspondence (e-mail: [email protected])

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The modular nature of flowering plant PcG complex compositionleads to the formation of individual PcG complexes, which facilitatethe control of different developmental programs along the plant lifecycle. Whereas PcG complexes containing either CLF or SWN actduring the sporophytic stage, for example in flowers and fruits(Chanvivattana et al., 2004; Katz et al., 2004), PcG complexescontaining either MEA or SWN act at the gametophytic stage, asevident from their mutant phenotype characterized by abnormalendosperm in the absence of fertilization and embryo abortion afterfertilization.

In view of the central role of the FIE PcG complex in regulatingthe transition of the female gametophyte to the sporophyte inflowering plants (Goodrich, 1998; Guitton et al., 2004; Kohler et al.,2003a; Ohad et al., 1996; Ohad et al., 1999), it is intriguing toanalyze the evolution of this function in basal land plants. For thispurpose, we chose the model bryophyte Physcomitrella patens, withits dominant gametophytic phase. Furthermore, different types ofstem cells can be analyzed in this plant, as the juvenile gametophyte(the protonema) is a filamentous tissue growing solely by apical celldivision, whereas the transition to the adult gametophyte (thegametophore) is characterized by a cell-fate transition to a three-faced apical cell (the bud). This transition can specifically betriggered by the plant hormone cytokinin (reviewed by Decker et al.,2006). Unique to land plants, reverse genetics approaches via genetargeting are highly efficient in P. patens (Reski, 1998b). In addition,the genome of P. patens has been entirely sequenced (Rensing et al.,2008), facilitating evo-devo studies with emphasis on the evolutionof specific transcription factors (Maizel et al., 2005; Sakakibara etal., 2008).

Here we show that P. patens FIE protein (PpFIE) accumulatesonly in gametophyte apical cells and cells that undergo fatetransition. Moreover, using targeted gene deletion and replacement,in the absence of PpFIE moss gametophore meristemsoverproliferate, but fail to develop and reach the reproductive phase,illustrating the key role of FIE in regulating proper differentiationand proliferation of P. patens gametophytic stem cells. This aberrantphenotype can be partially rescued by the FIE gene of A. thaliana,indicating functional conservation over more than 450 million years.Accordingly, PpFIE can partially complement the A. thaliana fiemutant. Thus the essential FIE PcG function in regulatingdevelopmental programs along the plant life cycle was establishedearly in evolution, around the water-to-land transition of plant life.

MATERIALS AND METHODSPlant material, culture conditions and treatmentsThe ‘Gransden 2004’ strain of P. patens (Ashton and Cove, 1977; Rensinget al., 2008) was propagated on BCD and BCDAT media (Ashton and Cove,1977) at 25°C under a 16-hour light and 8-hour dark cycle (Frank et al.,2005). All transgenic lines described in this study are deposited in theInternational Moss Stock Center with the accessions IMSC 40319-40324and 40265-40267 (PpFIE-GUS, ΔPpFIE- and AtFIE-co mutants).

Construction of the phylogenetic treeInitially, sequences for which BLAST hits were at least 30% identical overa length of 80 amino acids were selected in order to avoid inclusion of false-positive hits from the twilight zone of protein alignment (Rost et al., 1999).Only FIE homologs from organisms (plants and animals) for which thewhole genome sequence had been determined were taken into account. Anamino acid sequence alignment was generated using MAFFT G-INS-iversion 5.860 (Katoh et al., 2005) and manually curated using Jalviewversion 2.4 (Clamp et al., 2004). Based on the conserved core of thisalignment (essentially comprising several WD40 domains), a hiddenMarkov model was generated (HMMER 2.3.2; http://hmmer.janelia.org/), agathering cutoff of 400 was defined based on searches against several plant

and animal genomes, and this was subsequently used to detect and retrieveFIE homologs from a larger set of completely sequenced genomes. Basedon this set of sequences, a second alignment was constructed. The mostappropriate evolutionary model was selected using ProtTest version 1.3(Abascal et al., 2005) and turned out to be WAG (Whelan and Goldman,2001) with gamma-distributed rate categories. Bayesian inference (BI) wascarried out with the predetermined model using MrBayes version 3.1.2(Ronquist and Huelsenbeck, 2003) with eight gamma-distributed ratescategories (four chains, two runs) until convergence (average s.d.<0.01).Trees were visualized using FigTree version 1.2.2 (http://tree.bio.ed.ac.uk/software/figtree/) and manually rooted. Support values at the nodes (Fig. 1)are BI posterior probabilities. In addition, a neighbor-joining (NJ) tree wascalculated using QuickTree version 1.1 (Howe et al., 2002) with bootstrapresampling 1000 times. Neither the NJ nor the ProtTest maximum likelihoodtree was found to be significantly different from the BI tree.

Accession numbers are as follows: PpFIE, Phypa_61985 (PhyscomitrellaPatens); AtFIE, AAD23584 (Arabidopsis thaliana); ARALY_898508(Arabidopsis lyrata); EED, AAB38319 (Mus musculus); EED, AAH47672(Homo sapiens); CAG31770 (Gallus gallus); AAV36839 (Drosophilamelanogaster); Esc2, AAA86427 (D. melanogaster); EED, BAD22546(Oryzias latipes); AAH93351, LOC550463 (Danio rerio); POPTR_688045(Populus trichocarpa); FIE1, Os08g04270.1 (Oryza sativa); FIE2,Os08g04290.1 (O. sativa); XENTR_293769 (Xenopus laevis);FUGRU_713547 (Takifugu rubripes); OSTLU_37673 (Ostreococcuslucimarinus); OSTTA_22117 (Ostreococcus tauri); PHATR_9860(Phaeodactylum tricornutum); THAPS_118885 (Thalassiosirapseudonana); CHLRE_193732 (Chlamydomonas reinhardtii);CYAME_CMK173C (Cyanidioschyzon merolae); Selmo1_2_143777(Selaginella moellendorffii); VITVI_A7P0Y9 (Vitis vinifera);ZEAMA_148924 P.01 (Zea mays1); ZEAMA_118205 P.01 (Z. mays2)(Danilevskaya et al., 2003); ZEAMA_006312 P.01 (Z. mays3);RICCO_28166 (Ricinus communis); FIE1, GLYMA_10g02690.1 (Glycinemax); FIE2, GLYMA_02g17110.1 (G. max); FIE3, GLYMA_13g36310.1(G. max); FIE4, GLYMA_12g34240.1 (G. max); CARPA_7.67 (Caricapapaya); FIE1, SORBI_4986219 (Sorghum bicolor); FIE2,SORBI_4838275 (S. bicolor); NEMVE_102199 (Nematostella vectensis);VOLCA_58949 (Volvox carteri); MICP1_49065 (Micromonas pusilla);CHLSP_19370 (Chlorella sp. NC64A).

Gene isolationThe PpFIE and PpCLF complete coding regions were amplified fromcDNA by PCR using primers based on the ‘Joint Genome Institute’ (JGI) P.patens version 1.1 database (http://www.jgi.doe.gov) and were subsequentlycloned and sequenced. PpFIE turned out to be identical to Phypa_61985 (asavailable on www.cosmoss.org, version 1.2). The cDNA of PpCLF has beensubmitted to GenBank (accession: bankit1178009 FJ917288,www.ncbi.nlm.nih.gov), as no appropriate gene model is present at thegenomic locus (scaffold_100:687012-693616) in genome versions 1.1 and1.2. PpFIE is encoded by 1089 bp organized in a single exon, whereasPpCLF is encoded by 3000 bp spliced from 19 exons.

Construction of transformation vectorsPpFIE coding sequence (1089 bp) was amplified using the following primerset: PpFIE-Fw-1, 5�-AAGCTTCTCGAGATG GGAGATCT TGTCCCGG -ACAAG-3�; and PpFIE-Rev-1, 5�-AAGCTTTCAGC TAGCAGAC -ACAGCATCCCAGCGCCAAAT-3�. In addition, the 5� (805 bp) and 3�(598 bp) of the untranslated region (UTR) of PpFIE were amplified usingthe following primers: PpFIE5�-UTR-Fw, 5�-GAAGCTTG ACTA GAG -CAAAAAAATTGTGATAGTGTGT-3�; PpFIE5�-UTR-Rev, 5�-GAAG -CTTCACGGGATCCGTGCCGA-3�; PpFIE3�-UTR-Fw, 5�-GCATG -CTGAT CGTGGATATCTGGAGCCA-3�; and PpFIE3�-UTR-Rv, 5�-GCATGCCGCTAGGGTCATAGCCATATAAACA-3�. All amplifiedfragments were subcloned into the pTZ 57 vector (Fermentas, Lithuania)and sequenced to ensure their integrity. The PCR-amplified PpFIE genomicsequence was used for constructing the disrupted vector by the insertion ofthe selection cassette at the BalI site. The same genomic fragment wascloned in-frame to the uidA reporter gene at the NheI site to obtain a proteinfusion between PpFIE and GUS, and then cloned into the pMBL5 vector,

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followed by a nopaline synthase polyadenylation signal (NOS-ter), a nptIIcassette, as described in (Nishiyama et al., 2000), and the PpFIE3�-UTR (seeFig. S1 in the supplementary material). AtFIE full-length cDNA wasamplified using the primers AtFIE-F 5�-CCCGGGATGTC GAAG -ATAACCTTAGGG-3� and AtFIE-R 5�-CAAGGTCG ACG GGAGTA -GCAACAT-3�. The AtFIE cDNA was cloned into pMBL5 vector flanked byPpFIE5�-UTR at the 5� end and by the NOS terminator, the nptII cassetteand PpFIE3�-UTR, respectively, at the 3� end. Prior to transformation thevectors were linearized.

Protoplast isolation and PEG-mediated transformation ofP. patensPEG transformation was performed as described in PHYSCObase(http://moss.nibb.ac.jp). Six days after regeneration, transformants wereselected on BCDAT medium containing 20 mg/l of G418.

RT-PCRTotal RNA extraction from protonemata or leaves followed by RT-PCR wereperformed as described (Katz et al., 2004), using the following gene-specificprimers: for PpFIE, PpFIE-Fw-1 and PpFIE-Rev-1; for AtFIE, AtFIE-F andAtFIE-350-R 5�-GATGCTCGTTTCTTCGATGT-3�.

Real-time PCR analysis of gene expressionReal-time quantitative PCR analysis was performed by ΔΔCt method ofrelative quantification with a StepOne Thermal Cycler (Applied Biosystems,Foster City, CA, USA), using SYBR Green to monitor dsDNA synthesis.The following primers were used to detect PpFIE and the housekeepinggenes: PpFIE-left-261-278, 5�-AGATGGCAACCCCTTGCT-3�; PpFIE-Right-302-320, 5�-CAATCAATGATGCGGAGGA-3�; 60s-Left, 5�-GGGAACACTATCTTTTTCCTGGT-3�; 60s-Right, 5�-TGAAATCAT -GCGATTAGTCCTC-3�; TATA-Left, 5�-GATCTAGCTAT AAGCCTGAT -CTACCG-3�; TATA-Right, 5�-CAGGAGCAGGGAGAGATTTG-3�. Theamount of cDNA for each gene was quantified using a log-linear regressioncurve of the threshold cycle and the amount of standard template preparedfrom a cDNA clone.

Identification of A. thaliana fie mutant allele via RFLP analysisA. thaliana fie alleles carry a single point mutation resulting in apolymorphic site recognized by the DraI restriction enzyme. PCR analysiswas carried out on genomic DNA as a template using the primers AtFIE-2277-F 5�-GCTTGTGGTTCGTTTGTATG-3� and AtFIE-3143-R 5�-CCTATATGGCAACAGAAAAT-3� followed by restriction with the DraIenzyme, which enabled us to distinguish between the wild-type and mutantalleles. The resulting wild-type fragment was 866 bp, and the mutant gaverise to two products of 492 bp and 374 bp.

GUS assayThe histochemical assay for GUS activity was performed as described byNishiyama et al. (Nishiyama et al., 2000). The incubation time was adjustedfrom 2 to 24 hours, depending on the tissues examined.

Electron microscopyCryoscanning electron microscopy samples were frozen in liquid nitrogenon a copper sample holder, sputtered with 20 nm gold particles andvisualized using a JEOL 6300 cryoscanning electron microscope.Environmental scanning electron microscopy was performed using theQuanta 200 FEG with a field-emission gun and a gaseous secondary electrondetector for surface imaging in wet mode.

Bimolecular fluorescence complementation analysisProtein-protein interactions in plants were examined by bimolecularfluorescence complementation (BiFC) assay. PpFIE, PpCLF, AtFIE,AtMEA, AtCLF and AtSWN full-length cDNAs were cloned into pSY 735and pSY 736 vectors at the SpeI site, which contain the N-terminal (YN)and the C-terminal (YC) fragments of the YFP protein, respectively(Bracha-Drori et al., 2004). Equal concentrations of Agrobacteriumtumerfaciens strain GV3101/pMp90 containing plasmids of interest (seeTable 1) were transiently coexpressed in N. benthamiana leaves via the leafinjection procedure (Bracha-Drori et al., 2004). Image annotation wasperformed with Zeiss AxioVision, Zeiss CLSM-5 and Adobe Photoshop7.0 (Mountain View, CA, USA). The expression of each construct wasverified by its ability to interact with AtFIE (see Fig. S4G-I in thesupplementary material). Negative controls with vectors bearing only YNor YC alone were carried out in every experiment to verify the specificityof the interactions.

RESULTSThe FIE sequence is highly conserved among theeukaryotic crown kingdomsPutative homologs of A. thaliana FIE were collected using hiddenMarkov model (HMM) searches from organisms for which thewhole genome sequence had been determined. The phylogeny of theFIE protein superfamily is presented in Fig. 1. Potential FIEhomologs are also present in the genomes of organisms, includingC. elegans and Saccharomyces cerevisiae; however, their lowconservation grade does not allow for unambiguous assignment tothe FIE superfamily. It is evident from the phylogenetic tree that FIEis essentially a single-copy ortholog that was already present in thelast common ancestor of all eukaryotes and might subsequently havebeen lost in some (unicellular) lineages. The FIE phylogenyapproximately reflects the taxonomic relationships of the speciesinvolved. Paralog retention occurred occasionally and relatively lateduring evolution (after the insect-vertebrate split and the monocot-eudicot split, as can be seen from the D. melanogaster, O. sativa, Z.mays, S. bicolor and G. max paralogs, Fig. 1). The high conservationof the FIE proteins (e.g. 66% identity, 81% similarity over the wholeprotein length between A. thaliana and P. patens and 41% identity,

2435RESEARCH ARTICLEFIE PcG protein regulates stem cell development

Table 1. Vectors used in this studyVector Description Source

pMBL5 SK backbone with nptII cassette GenBank accession number DQ228130pTZ 57 TA cloning vector Fermentas, LithuaniapTZ ΔPpFIE pTZ PpFIE genomic nptII (BalI) This studypMBL5 PpFIE GUS pMBL PpFIE GUS NOS nptII 3� UTR This studypMBL5 AtFIE pMBL cDNA AtFIE NOS nptII 3� UTR This studypCmbia 4.2 P:PpFIE pCambia 4.2 AtFIE promoter:PpFIE This studypSY 736 YN only (for negative control) (Bracha-Drori et al., 2004)pSY 735 YC only (for negative control) (Bracha-Drori et al., 2004)pSY 736-AtFIE YN-MSI1 (Bracha-Drori et al., 2004)pSY 736-AtMEA YN-AtMEA (Bracha-Drori et al., 2004)pSY 736-AtCLF YN-AtCLF This studypSY 736-AtSWN YN-AtSWN This studypSY 736-PpFIE YN-PpFIE This studypSY 735- PpFIE YC-PpFIE This studypSY 736-PpCLF YN-PpCLF This studypSY 735-PpCLF Yn-PpCLF This study D

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60% similarity between A. thaliana and H. sapiens) across themillennia underlines its conserved structure and indicates its crucialfunction for higher eukaryotes (Fig. 1).

PpFIE-GUS accumulates in all meristematic cellsand gametophytic cells undergoing fatetransitionTo determine PpFIE temporal and spatial accumulation during P.patens development, we generated five transgenic plants in whichthe uidA (GUS) reporter gene was inserted via homologousrecombination, replacing the PpFIE stop codon. The resulting plantsexpress a PpFIE-GUS fusion protein under control of its nativepromoter within the endogenous genomic environment.

The PpFIE-GUS staining pattern was identical among all fivemoss transgenic lines generated. None of these lines exhibited anyobvious abnormalities as compared with wild-type plantmorphology, nor were any changes detected in the course and timingof their life cycle. Therefore, we conclude that PpFIE function fordevelopmental control in PpFIE-GUS plants was not impaired.

During the life cycle of wild-type P. patens, the fertilized zygotedevelops into a sporophyte consisting of a reduced seta and the sporecapsule. Several days after spore dispersal, the wild-type haploidspores germinate forming the juvenile gametophyte, a branchedfilamentous tissue growing by apical cell division. This protonematissue comprises two subsequently occurring cell types, thechloronemata and the caulonemata. The division of the apical cellproduces protonemal filaments, whereas subapical cells may divideto produce either side branch initials or three-faced apical cells, thebuds. These buds subsequently develop into the adult gametophyte,the leafy shoots (gametophores) that bear the sex organs (Cove et al.,2006; Cove and Knight, 1993; Decker et al., 2006; Reski, 1998a;Schaefer and Zryd, 2001).

No FIE was detected by GUS staining in the spores afterdispersal, but the protein appeared during imbibition before sporegermination (Fig. 2A,B). Upon germination (Fig. 2B,C) andthroughout the protonema phase, a weak GUS staining was detectedin the apical cells of caulonemata and chloronemata (Fig. 2D,E).

A strong GUS staining was visible at the time of transition fromthe juvenile to the adult gametophyte, marked by the transition tothree-faced apical cells, the buds (Fig. 2F). Subsequently, strongGUS staining was consistently detected in the apical and lateralshoot apices of the leafy gametophores (Fig. 2G-J). In the course ofthe GUS staining process, the apical cell of the gametophore stainedfirst, followed by the adjacent cells (Fig. 2I,H). During gametophoredevelopment, PpFIE-GUS staining decayed gradually from theapical cell towards differentiated leaves (Fig. 2H). Throughoutlateral shoot formation, the PpFIE protein could be monitored asearly as in a single cell designated to form a lateral shoot (Fig. 2J,arrowheads).

Upon transition from the gametophyte to the reproductivephase, GUS signals were associated with the male and femalereproductive organs antheridia and archegonia, respectively.Before fertilization, GUS activity was detected in the developingarchegonia (Fig. 2K,L), where staining was particularly strong inthe unfertilized egg cell (Fig. 2L, see arrow), whereas the signalgradually decayed after fertilization (Fig. 2M-O). Likewise, GUSsignals were also evident in the young antheridia carrying thespermatozoids (Fig. 2P), whereas no signal was detected uponsperm maturation (Fig. 2Q). After fertilization no GUS signal wasdetectable, neither in the developing embryo, nor in the maturesporophyte until the stage of spore formation (Fig. 2R). Here,GUS staining was only found during spore formation in thetetrads after meiosis (Fig. 2S) and was undetectable in maturespores (Fig. 2T).


Fig. 1. Phylogenetic tree (Bayesian inference) of the FIE protein superfamily. Numbers at the nodes represent posterior probabilities. The treewas rooted at the branch separating multicellular animals from plants and algae. Only clear true-positive homologs from organisms for which thewhole genome sequence had been determined were included. In the case of paralogy the proteins are labeled using index numbers.

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PpFIE-GUS protein accumulation patterns reveal that the proteinis present in all meristematic cells and gametophytic cells undergoingfate transition. The complex pattern of PpFIE-GUS localizationsuggests that it is involved in various developmental processesrestricted to the haploid tissue during the P. patens life cycle.

ΔPpFIE mutants overproliferate three-faced apicalcellsTo study PpFIE function during P. patens development, wegenerated four independent disruptant mutant lines, designatedΔPpFIE, by gene targeting via homologous recombination (see Fig.S1A in the supplementary material). Proper integration of thedisrupting construct into the PpFIE locus was verified byamplifying and sequencing the junction sites between the insert andthe PpFIE locus (see Fig. S1A in the supplementary material).Single event of integration was determined by Southern blotanalysis (see Fig. S6 in the supplementary material) and completeloss of PpFIE transcripts due to the disruption was confirmed byRT-PCR analysis (see Fig. S2A in the supplementary material).Subsequently, the phenotype of the ΔPpFIE mutants was monitoredalong with their development. ΔPpFIE protonemata and bud initialsappeared indistinguishable from those of wild type (Fig. 3A,F).However, during the transition from juvenile to adult gametophytes,marked by the transition from apical cells to three-faced apical budinitials, ΔPpFIE mutants displayed dramatic morphologicalalterations (Fig. 3B,G). Whereas wild-type protonema gave rise tobuds that further developed into leafy gametophores (Fig. 3B-E),ΔPpFIE developed a mass of cone-shaped buds (Fig. 3H, insertion)that grow further, thus harboring multiple apices (Fig. 3G-J). Thesemutant buds developed into cone-like leafless gametophores (Fig.3J), whereas cones remote from the main apex initiated thedifferentiation of leaf primordia (Fig. 3J, insertion). Each main budcontinued to repeatedly produce additional successive orders ofprimordia, until numerous apices accumulated on the main budsurface (Fig. 3I,J).

This aberrant phenotype indicates that ΔPpFIE mutant plants failto restrict bud proliferation, resulting in ectopic initiation anddifferentiation of multiple bud apices. Furthermore, these buds failedto mature or to form normal gametophores and thus are preventedfrom reaching the reproductive phase. However, as some of themutated structures resembled the morphology of a sporophyte (Fig.3H, insertion), we tested whether such structures acquiresporophytic identity. To this end we have tested the expression of thegenes MKN2 and MKN5, which were shown to be expressedspecifically in sporophytic tissue (Sakakibara et al., 2008). Ourresults show that MKN2 and MKN5 are expressed in ΔPpFIEprotonemata bearing abnormal buds but not in wild-typeprotonemata bearing gametophores (Fig. 3N).

PpFIE is associated with the maintenance ofpluripotency and cell reprogrammingP. patens cells have remarkable regenerative plasticity followingtissue damage (Cove and Knight, 1993), indicating that cells retaintheir capability to exit their determined state after differentiation.During P. patens regeneration, all differentiated cells undergodivision, giving rise to protonemata. As our results indicate thatPpFIE is involved in maintaining the undifferentiated state of apicalcells, we examined whether PpFIE protein accumulation correlateswith the regeneration process. To induce this process, leaves fromPpFIE-GUS transgenic lines were detached from maturegametophores and placed on BCD media for different periods oftime up to 88 hours and subsequently stained for GUS (Fig. 4).Following detachment, we monitored the regeneration process at thesurface of the distal leaf region, rather than the proximal marginalregion of the incision site where wounding might affect FIEexpression. Forty-eight hours after induction, single scattered cellsexpressing PpFIE-GUS were observed on the leaflets (Fig. 4C,I). At72 hours some of the GUS-expressing cells were observed to


Fig. 2. PpFIE-GUS protein expression pattern determined byhistochemical GUS assays. Analysis of PpFIE-GUS lines.(A-C,E) Germinating spores, arrow indicates the apical protonema cell.(D) The protenema tip. (F) A juvenile bud. (G-J) Leafy gametophore (G)and a subtending meristematic cell (J) exposed after removal ofsurrounding leaves. The apices of the main (arrow) and lateral shoots(arrowhead) are indicated in G and J. Staining of the apical cell; 5 hoursin H and 2 hours in I. (K-O) A developing archegonia. GUS activity wasdetected in the egg cell as well as in the archegonia tissue; arrow in Lmarks the egg cell. (P,Q) Antheridia. (R,T) A sporophyte. (S) Sporeformation; box shows tetrad after meiosis. Scale bars: 25μm in A-C;50μm in I-Q,S; 100μm in R,T,E; 1 mm in G.

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undergo cell division (Fig. 4K, arrows). After 88 hours protonemafilaments emerged from cells in which GUS staining was visible(Fig. 4L, arrowhead).

In order to examine whether PpFIE transcription is upregulatedduring the regeneration process, wild-type leaves were detached andincubated as described above. RNA was purified fromapproximately 1000 leaves collected at time ‘0’ and 72 hours afterdetachment. Quantitative RT-PCR analysis showed that the FIEtranscript is upregulated at least twofold 72 hours after leafdetachment, as compared with time ‘0’ (Fig. 4M).

PpFIE upregulation and the spatial and temporal pattern ofaccumulation of the protein during the regeneration process indicatethat the epigenetic machinery is involved during cell reprogrammingand acquisition of pluripotency.

A. thaliana FIE is able to complement P. patens FIETo determine whether FIE protein function is conserved betweenbryophytes and angiosperms, we performed a cross-speciescomplementation assay. To this end, transgenic P. patens lines weregenerated, in which the endogenous PpFIE was replaced viahomologous recombination with an AtFIE cDNA driven by thenative moss PpFIE promoter (designated AtFIE-co). Fourindependent P. patens AtFIE-co lines were isolated expressing

AtFIE (see Fig. S1B and Fig. S2B in the supplementary material).All four AtFIE-co lines partially complemented the lack of theendogenous PpFIE (Fig. 3K-M, Fig. 5B). Similar to ΔPpFIE mutantlines, AtFIE-co lines exhibited abnormal buds with multipleprimordial leaves at the early phase of growth (Fig. 3K). However,in contrast to ΔPpFIE plants, AtFIE-co lines were able to developmature gametophores (Fig. 3L,M), although AtFIE-co linesexhibited distinct abnormal phenotypes. The distance betweenleaves along the gametophore was shorter, resulting in a denserappearance as compared with wild-type gametophores (compareFig. 5A and 5B). At the apex of mature gametophores, clusters ofshoot apices developed, bearing juvenile leaves (Fig. 5C, arrows).This phenomenon was also observed in lateral shoot apices (data notshown). Under reproduction-inducing conditions (Hohe et al.,2002), AtFIE-co lines failed to produce sex organs. The PpFIE-GUSaccumulation pattern overlapped with the sites in which the abovementioned abnormalities were observed. The failure of AtFIE tosupport the proper development of reproductive organs could be dueto the inability of AtFIE to recognize additional subunits present inthe PcG complex at this particular P. patens developmental stage.These results demonstrate that FIE has been functionally conservedthrough evolution, thus allowing partial rescue of the ΔPpFIEmutant using AtFIE.


Fig. 3. Morphological analysis of wild-type, ΔPpFIEand P. patens plants in which PpFIE is replaced bythe AtFIE gene. Wild-type (A-E) and ΔPpFIE (F-J)plants, and P. patens plants containing AtFIE instead ofPpFIE (K-M). (A,F) Light microscopy of juvenile budconsisting of few cells. (B) Bud with leaf primordia.(G)ΔPpFIE bud bearing multiple apices. Two-week-oldcolonies of wild type (C), ΔPpFIE (H) and AtFIE-co (K)grown on BCD media. Insertion in H is magnified view.Color images were taken by light stereomicroscopy.CryoSEM images of wild-type bud (D), ΔPpFIEoverproliferating buds (I,J) and a mature bud of AtFIE-co(L). Wild-type (E) and AtFIE-co (M) gametophore.(J) Mature ΔPpFIE overproliferating buds. (N) RT-PCRanalysis of MKN2 and MKN5 gene expression in wildtype and ΔPpFIE, as compared with PpGAPC1 levels tomonitor input amounts. Scale bars: 100μm in A,B,E,F,G-J,L,M; 1 mm in C,H,K; 50μm in D; 200μm in insertionin H.

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PpFIE partially complements the gametophyticlesion of the A. thaliana fie mutantThe ability of AtFIE to rescue P. patens plants lacking PpFIEindicates that AtFIE can functionally recognize components of theP. patens PcG complex. Thus we performed the reciprocalexperiment and analyzed whether PpFIE can functionallycomplement the absence of AtFIE in the A. thaliana PcG complex.

To this end, we tested whether PpFIE can rescue the abortedembryo of A. thaliana plants in which the female gametophytecontributes a fie mutant allele (Ohad et al., 1996; Ohad et al., 1999;Chaudhury et al., 1997). To test this possibility we established sixindependent transgenic lines expressing PpFIE under the A. thalianaFIE native promoter (ProAtFIE:PpFIE) as described by Kinoshitaet al. (Kinoshita et al., 2001). To establish the complementationassay, we first crossed ProAtFIE:PpFIE A. thaliana lines as femalerecipients with pollen from heterozygous FIE/fie plants. F1progenies from the six parents were selected for kanamycinresistance as an indication for the presence of the ProAtFIE:PpFIEtransgene. These lines were than screened for the presence of the fieallele as determined by seed abortion (Ohad et al., 1996). In addition,the same heterozygous F1 plants (FIE/fie) hemizygous for thetransgene (PpFIE/~; plant genotype designated as FIE/fie,PpFIE/~)were tested by DNA restriction analysis for the presence of the fiemutant allele, monitoring for the unique DraI polymorphic site (seeFig. S3C in the supplementary material). The expression of thePpFIE transgene in these lines was confirmed by RT-PCR analysis(see Fig. S3B in the supplementary material). F2 progenies werecollected and germinated, out of which FIE/fie,PpFIE/~ plants wereselected as described above. Complementation was assessed byscoring F3 progeny seed abortion ratio in siliques from individualF2 plants, derived from self pollination.

In the case that PpFIE would fully complement the fie mutantallele, one would expect that F2 plants carrying both alleles woulddisplay 25% seed abortion in the F3 generation (Ohad et al., 1999),in contrast to 50% seed abortion if no complementation occurs. Outof six independent lines, two displayed abortion of approximately45% (Table 2, rows 1 and 2), which is significantly lower than 50%(χ2 test, P<0.001), in which the morphology of aborted embryos wasnot different from FIE/fie aborted embryos. These results indicatethat the bryophyte FIE protein partially complements for the absence

of a functional FIE allele in the flowering plant female gametophyte,thus supporting early seed development and allowing embryorescue.

An additional genetic approach was employed to determinewhether PpFIE is able to complement the A. thaliana fie allele.The A. thaliana fie mutant allele causes embryo lethality whentransmitted through the female parent, with 100% penetration(Ohad et al., 1996). Thus, the fie mutant allele is not transmittedby the female parent but only through the male. A cross betweena FIE/fie female with a wild-type male will result in 100% wild-type F1 plants (Table 2, row 6). However, if PpFIE cancompensate for A. thaliana fie lack of function in the female


Fig. 4. PpFIE expression and protein accumulation in detached, somatic leaves. GUS staining of detached leaves from the gametophore,incubated in BCD medium for time intervals of 24, 48, 57, 72 and 88 hours. (A-F) PpFIE-GUS staining of gametophore leaves. (G-L) Magnificationof the respective images. Dividing cells are marked with an arrow, differentiated protonema grown on the leaf in L is marked with an arrowhead.(M) Quantitative RT-PCR analysis of PpFIE expression at time ‘0’ and 72 hours after leaf detachment. Scale bars: 0.1 mm.

Fig. 5. Morphological analysis of mature gametophores fromwild type and plants in which PpFIE is replaced by the AtFIEgene. (A) Wild-type adult gametophore. (B) Adult AtFIE-cogametophore exhibiting clusters of shoot apices bearing juvenile leavesat the shoot apex. (C) Magnification of an AtFIE-co apex (box in B).Arrows mark the leaflets that formed on the proliferated apex. Theimages were taken by light stereo-microscopy. Scale bars: 1 mm in A,B;0.1 mm in C. D

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gametophyte, then a heterozygous FIE/fie female carrying thePpFIE transgene is expected to transmit the A. thaliana fie mutantallele to the next generation.

F1 progeny resulting from a cross between aFIE/fie,PpFIE/~female and a wild-type male (see Fig. S4A,B,column P and Fig. S4C,D in the supplementary material) carrying aGFP marker were screened by RFLP analysis for the presence of thefie mutant allele (see Fig. S4A,B, column F1 in the supplementarymaterial). In addition, progenies obtained from self-fertilization ofthe above F1 plants were tested morphologically for seed abortion(see Fig. S4E in the supplementary material). Out of 67 F1progenies, four carried the fie allele, whereas in the controlexperiment in which a FIE/fie female was crossed with the samewild-type male donor, all 192 resulting plants were homozygous forthe FIE wild-type allele as expected. The presence of the GFPmarker provided by the male donor assured that the outcrossingoccurred properly.

Although PpFIE was able to facilitate the transmission of thefemale A. thaliana fie allele, none of the F3 progenies in thecomplementation experiment was homozygous for A. thaliana fie.

Since the paternal AtFIE allele is apparently not expressed until lateembryogenesis (Yadegari et al., 2000), we suggest that under theexperimental conditions we have used PpFIE complementation isdelimited to the early phase, during which PpFIE is providedmaternally.

Based on our findings that PpFIE can partially complement the A.thaliana fie lesion at particular developmental stages, and that AtFIEcan replace PpFIE at the bud stage, we conclude that there has beenpartial functional conservation of FIE during land plant evolution.

PpFIE and PpCLF PcG proteins interact in plantaIn A. thaliana, FIE and SET domain proteins were shown to interactdirectly (Katz et al., 2004), as in the case for the homologous PcGproteins in D. melanogaster, Mus musculus and H. sapiens(reviewed by Berger and Gaudin, 2003; Hsieh et al., 2003; Simonand Tamkun, 2002). To test whether PpFIE and PpCLF are able tointeract, as expected from their proposed function, we used the BiFCassay (Bracha-Drori et al., 2004). To this end we cloned the full-length cDNAs of PpFIE and PpCLF, each fused to either the N-terminal (YN) or C-terminal (YC) fragments of the YFP encoding


Fig. 6. PpFIE and AtFIE interact with PcG SET domain proteins in planta. BiFC analysis of in planta interactions between YC-PpFIE and thefollowing: YN-PpCLF (A), YN-AtCLF (B), YN-AtMEA (C), YN-AtSWN (D) and YN-only as a negative control (E). As a positive control YC-AtFIE wastested with YN-PpCLF (F), YN-AtCLF (G), YN-AtMEA (H), YN-AtSWN (I) and YN-only as a negative control (J). Negative controls examining YC-onlywith YN-PpCLF (K), YN-AtCLF (L), YN-AtMEA (M) and YN-AtSWN (N). Localization was determined in the leaf epidermis of Nicotiana benthamiana.YFP fluorescence from single confocal sections is overlaid with Nomarsky differential interference contrast (DIC) images. Scale bars: 50μm.

Table 2. Abortion rates of wild type, A. thaliana fie mutants and A. thaliana fie mutants complemented with PpFIE

Genotype Abortion ratio Abortion average ratio χ2 P value/H0

PpFIE/~,FIE/fie line 1 469:586, 1:1 44.45 13.00 0.003 (50%)*PpFIE/~,FIE/fie line 17 611:728, 1:1 45.59 10.4 0.001 (50%)*FIE/fie 879:804, 1:1 52.22 3.34 0.068 (50%)PpFIE/~,FIE/FIE 3:1162, 0:1 0.002 – –FIE/FIE – wild type 3:1328, 0:1 0.002 – –F1 of FIE/FIE female � FIE/fie male 2:880, 0:1 0.002 – –

χ2 test was used to determine whether the H0 hypothesis of 50% seed abortion fit with the different genetic categories. Lines marked with * exhibit a significantly lowerstatistical rate of seed abortion than the expected 50%. D

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sequence. Members of each protein pair were transientlycoexpressed via Agrobacterium tumefaciens-mediatedtransformation in leaf epidermal cells of Nicotiana benthamiana.YFP fluorescence was observed in cells expressing both YC-PpFIEand YN-PpCLF (Fig. 6A). No fluorescence was observed whenexpressing YC-PpFIE with YN only (Fig. 6E) or YN-PpCLF withYC only (Fig. 6K), both serving as negative controls. Interactionbetween both proteins was localized in the nucleus, which is inagreement with the known functions of the PcG in regulatingchromatin structure (Dingwall et al., 1995; Francis et al., 2004; Paroand Hogness, 1991). The above result supports the hypothesis thatPpFIE and PpCLF interact in vivo to form the core of a PcGcomplex.

The complementation tests described above suggest that both P.patens and A. thaliana FIE proteins interact with the respective PcGcomplex subunits in the other species. We next tested whether FIEproteins from either P. patens or A. thaliana were able to interactwith their counterpart SET domain proteins. Using the BiFC assaywe show that PpFIE can interact with AtCLF and AtSWN (Fig. 6Band 6D), but fails to interact with AtMEA (Fig. 6C). All interactionsoccurred in the nucleus. Whereas the interaction between PpFIE andPpCLF gave rise to a reconstitution of the YFP in almost all cells,the interaction with AtSWN appeared only sporadically. As negativecontrols, neither the SET domain proteins nor PpFIE or AtFIEinteracted with the half-complementing counterpart YFP proteinalone (Fig. 6K-N,E,J, respectively). In addition, we observed aninteraction between AtFIE and PpCLF (Fig. 6F). These cross-species protein interactions are in agreement with the geneticcomplementation assays described above. P. patens FIE couldinteract only with A. thaliana CLF or SWN, which are SET domainproteins that have evolved earlier during plant evolution comparedwith AtMEA, the most recently derived gene among the A. thalianaSET domain family (Spillane et al., 2007).

DISCUSSIONPcG function has been conserved during plantevolutionThe Polycomb group (PcG) complex controls gene expressionprofiles epigenetically. In this study we identified P. patens single-copy orthologs to the A. thaliana PcG complex core (FIE) and SETdomain catalytic (CLF) subunits. Two lines of evidence indicate thatPpFIE is a true functional ortholog of the PcG core subunit. First,even though the reciprocal complementation assays between P.patens and A. thaliana were limited to specific developmentalstages, they demonstrate that the FIE genes have maintained theirfunction during evolution. Second, our BiFC experiments show thatPpFIE interacts with two A. thaliana SET domain PcG proteins,AtCLF and AtSWN.

Further studies will reveal the extent of functional conservationof PpFIE as a member of a transcriptional repressor complex(PRC2) alongside the A. thaliana life cycle. To this end, the abilityof PpFIE to regulate AtFIE target genes, evident by either ChIPassay or the analysis of marker genes in vivo, could be applied.

Interestingly, PpFIE did not interact with AtMEA, which is themost recently diverged member in the SET domain protein familypresent in flowering plants (Chanvivattana et al., 2004; Spillane etal., 2007). Although all three A. thaliana SET domain proteins areexpressed in the ovule (Chanvivattana et al., 2004; Goodrich et al.,1997; Grossniklaus et al., 1998; Wang et al., 2006; Xiao et al., 2003),MEA has a more prominent function in regulating central celldevelopment (Grossniklaus et al., 1998; Kiyosue et al., 1999; Wanget al., 2006). The lack of interaction between PpFIE and AtMEA in

the BiFC assay agrees with the partial complementation in A.thaliana by PpFIE, which could result from the inability of PpFIEand AtMEA to form a complex in the central cell. Consistent withthis hypothesis, it was recently shown that the apomictic Hieracium(H. piloselloides) and non-apomictic Hieracium (H. pilosella) FIEproteins differentially bind members of the PcG complex, thuslimiting their later function. The inability to bind particular partnerswas attributed to specific modifications at the protein level, whichmay lead to structural changes between the two proteins (Rodrigueset al., 2008).

In mammals, the PcG complex exerts its function by methylatingH3K27 via the SET domain of the enhancer of zeste subunit(Czermin et al., 2002; Muller et al., 2002). Our analysis shows thatPpFIE and the SET domain protein PpCLF interact in vivo (Fig. 6),which supports the possibility that in P. patens these proteins forma complex to perform a similar biochemical function as in mammals.This hypothesis is supported by the conservation of the catalyticPpCLF SET domain protein in P. patens, as this is also present in A.thaliana, H. sapiens and D. melanogaster (see Fig. S5 in thesupplementary material).

FIE function in P. patensDuring wild-type bud formation the apical cell divides, giving riseto a subset of three-faced apical daughter cells. After severalconsecutive cell divisions some of the peripheral cells along thesurface of the young bud give rise to either leaf primordia(Schumaker and Dietrich, 1997) or meristematic cells thatsubsequently develop into lateral shoots. PpFIE-GUS protein levelsare correlated with organ differentiation, as they gradually declinefrom the bud apex towards the region where leaf initials emerge,until the fusion protein can no longer be observed. Bud initiation inΔPpFIE mutants is indistinguishable from wild type (Fig. 3A,F),thus PpFIE is not essential for the initiation of the three-faced apicalcell. However, soon after the ΔPpFIE three-faced apical cell givesrise to several daughter cells, secondary buds are initiated, formingmultiple apices in a repeatable pattern (Fig. 3G). These secondarybuds initiate leaf primordia only after they grow further apart fromthe center of the main bud cluster (Fig. 3J).

Taking the PpFIE protein expression pattern and ΔPpFIE mutantphenotype together, we hypothesize that in wild-type P. patens FIEfunctions to maintain an undifferentiated state of meristematic cellswithin the apex. These cells are designated to become lateral shoots,probably by the epigenetic repression of gene expression. Thisrepression can be relieved by as yet unknown signals, thus allowingthe initiation of lateral shoot formation, as the apex is pushedupwards by its daughter cells. Thus the ΔPpFIE mutant phenotypemight result from failure of the PcG complex to repress suchmeristematic cells from pursuing their default program todifferentiate into lateral buds in due time.

Whereas in the wild type each bud gives rise to individualgametophores bearing leaves, ΔPpFIE mutants develop multiplebud apices that fail to form leafy gametophores. However, mutantbuds continue to proliferate, until marginal buds generateundeveloped leaf primordia, thus being relieved from the repressedstate they were in. These results indicate that the potential to developleaves exists in these mutant apices (Fig. 3J, see insertion).

This phenomenon might not necessarily derive directly from theabsence of PpFIE. Rather, it might result from the presence ofmultiple apices, leading to either overproduction of a signalinhibiting leaf differentiation, or it might be due to the dilution of arequired signal consumed by the overproliferating buds situatedwithin close vicinity.


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The proposed role of PpFIE, as revealed from the ΔPpFIEphenotype and PpFIE expression pattern, is to maintain thepluripotency of the three-faced apical daughter cell. This fits wellwith the known function of PcG proteins during stem celldifferentiation (for reviews, see Kanno et al., 2008; Pietersen andvan Lohuizen, 2008). Mammalian EED and ESC PcG proteins,homologs of FIE and CLF, respectively, take part in the maintenanceof stem cells by preventing their differentiation (Boyer et al., 2006;Bracken et al., 2006; Lee et al., 2006). Mutant stem cells lackingEED cannot maintain their pluripotency and are prone todifferentiate (Boyer et al., 2006; O’Carroll et al., 2001). Similarly,in the A. thaliana fie mutant the central cell, serving as a stem cell,proliferates and differentiates precociously into juvenile endospermwithout fertilization (Chaudhury et al., 1997; Ingouff et al., 2005;Ohad et al., 1996; Ohad et al., 1999), demonstrating that the PcGcomplex represses the central cell from differentiating to endospermprior to fertilization.

Thus, the role of FIE in bryophyte development, as revealed inthis study, agrees with the basic function of the PcG complex toregulate self-renewal and inhibit differentiation. This developmentalrole is conserved in both the plant and the animal kingdom.

PpFIE function during reproductive developmentΔPpFIE mutants were unable to develop gametophores, thusremaining infertile. However, PpFIE-GUS was detected during sexorgan and gamete formation, yet was excluded from the zygote oncefertilization took place, implying that it has a possible functionduring sexual development. Furthermore, when replacing the nativePpFIE with AtFIE, the resulting transgenic plants were able todevelop gametophores. However, the apical cells of these plantsfailed to produce sex organs, and developed multiple apices on topof the gametophore apex instead. It is possible that at thisdevelopmental stage, AtFIE, in contrast to PpFIE, fails to recognizeparticular P. patens PcG subunits, which are crucial for properdevelopment. Taken together, the above results indicate that PpFIEtakes part in regulating the transition from the vegetative to thereproductive phase.

In support of the above we found that ΔPpFIE mutants formcone-like structures resembling young sporophytes. These mutantsalso express the sporophytic marker genes MKN2 and MKN5(Sakakibara et al., 2008). Thus, these results indicate that PpFIEcontrols the transition of particular developmental stages along theP. patens life cycle, including the transition from the gametophyticto the sporophytic stage. This function of the PcG complex has beenretained for more than 450 million years of plant evolution.

PpFIE function during redifferentiationPpFIE is present mainly in apical meristematic cells but is absentfrom cells that have already differentiated, such as in mature leaves.Our data show that PpFIE expression and protein accumulationprecede the regeneration processes in which leaf somatic cells areabout to regenerate, giving rise to protonema (Fig. 4), as well as inwounded tissue (data not shown), and further support the proposedfunction in establishing self-renewal and pluripotency. We predictthat leaf regeneration first requires de-differentiation of leaf cells,allowing them to pass through a meristematic state before they canacquire a new identity. Upregulation of PpFIE might allow the cellto enter into a reprogrammable state facilitated by chromatinremodeling, as expected from PcG function.

So far the analysis of plant cell de-differentiation andregeneration has been performed mainly with protoplasts (for areview, see Grafi, 2004). Our results now show that the PpFIE

protein might serve as a novel molecular marker, highlightingcells that are about to divide and differentiate, thus serving as atool to monitor these processes during the development of theentire plant and leaf regeneration.

Evolution of the PcG role in developmentPpFIE is mainly expressed in gametophytic tissues. The transitionof land plants from haploid dominancy to diploid dominancy mayhave evolved either by utilizing established developmental pathwaysacting within the gametophyte (the ‘homologous’ theory) or vianovel genes and networks that arose specifically to support thisprocess (the ‘antithetic’ theory) (Bennici, 2005). Support for thehomologous model was suggested in the case of PpRSL1 andPpRSL2, which control gametophytic rhizoid formation in P. patens,whereas their orthologs AtRSL1 and AtRHD6 control sporophyticroot hair development in A. thaliana (Menand et al., 2007). Ourresults show that the PRC2 epigenetic machinery, in which FIE isincluded, was maintained through the evolution of land plants,repressing the differentiation of meristematic cells in thegametophyte. This is supported by the observation that PpFIE-GUSwas detected only in haploid tissues.

During the evolution of land plants the PcG machinery wasrecruited to regulate the proper development of sporophyticprograms, such as transition from the vegetative to the reproductivephase, flowering time and flower organ formation (for reviews, seeGuitton and Berger, 2005; Hsieh et al., 2003; Kohler andMakarevich, 2006). This was accompanied by the diversification ofthe SET domain in P. patens to a gene family in A. thaliana, allowingthe formation of diverse PcG complexes, as seen in the case ofMEA, which is specialized in regulating endosperm development(Kawabe et al., 2007; Miyake et al., 2009; Spillane et al., 2007).

Thus, the findings described here highlight an example whereby,instead of harnessing pre-existing transcription factors, higher-orderepigenetic machinery was recruited to regulate the expression ofparticular gene sets to control evolving developmental programs.

AcknowledgementsWe thank W. Frank for help with the initial analysis of the FIE gene, M.Bodas for technical assistance, M. Panijel for assistance with Southern blotanalysis, and M. Oliva and I. Nevo for assistance in constructing the MEA andEZA BiFC vectors. We are grateful to the Center for Microscopy of theUniversity of Basel (Switzerland) and to Drs Z. Barkay (Wolfson Center) andV. Wexler (IDRFU), both at Tel Aviv University, for SEM analyses; D. Chamovitzfor critical reading; and Y. Butenko for graphical assistance. A.M. wassupported in part by a matching Tel Aviv University Deans DoctoralFellowship and the Manna Foundation. S.A.R. is grateful for funding by theExcellence Initiative of the German Federal and State Governments (EXC294, bioss). This research was supported by a grant from the German-IsraeliFoundation for Scientific Research and Development (GIF, 832-130) grantedto N.O., R.R. and E.L.D.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/14/2433/DC1

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